Generation of cryopreserved macrophages from normal and genetically engineered human pluripotent stem cells for disease modelling

Macrophages are innate immune cells that play critical roles in tissue homeostasis, inflammation, and immune oncology. Macrophages differentiated from human induced pluripotent stem cells (iPSCs) overcome many limitations of using peripheral blood derived macrophages. The ability to scale up and cryopreserve a large amount of end stage macrophages from single clonal iPSCs from normal and disease specific donors offers a unique opportunity for genomic analysis and drug screening. The present study describes the step wise generation and characterization of macrophages from iPSCs using a defined serum free method amenable to scale up to generate a large batch of pure end stage cryopreservable macrophages expressing CD68, CD33, CD11c, CD11b, CD1a, HLA-DR, CD86, CD64, CD80, CD206, CD169, CD47, HLA-ABC, and CX3CR. The end stage macrophages pre and post cryopreservation retain purity, morphology, responsiveness to stimuli and display robust phagocytic function coming right out of cryopreservation. The same differentiation process was used to generate end stage macrophages from isogenic iPSCs engineered to mimic mutations associated with Parkinson’s disease (SNCA A53T), neuronal ceroid lipofuscinosis (GRN2/GRN R493X), and Rett syndrome (MECP2-Knockout). End stage macrophages from isogenic engineered clones displayed differential macrophage-specific purity markers, phagocytic function, and response to specific stimuli. Thus, generating a panel of functional, physiologically relevant iPSC-derived macrophages can potentially facilitate the understanding of neural inflammatory responses associated with neurodegeneration.


Introduction
Macrophages are found in various tissues throughout the body and display a diverse array of morphological and physiological functions. Macrophages are derived from the myeloid lineage and are involved in not only the innate immune system, but also play a crucial role in cell Alpha-synuclein, a 140 amino-acid protein, accounts for about 1% of the total cytosolic proteins in the brain. Its expression is highest in the dopaminergic neurons and is intracellularly localized in presynaptic terminals. Alpha-synuclein proteins can self-assemble, passing from unfolded monomers to oligomeric species, and then to heavy aggregates (called amyloid fibrils). The accumulation of these insoluble fibrils progressively promotes the formation of intracellular inclusions called Lewy bodies within neurons and glial cells [10]. Most of PD cases are sporadic point mutations of SNCA, the gene encoding the Alpha-synuclein protein, which cause inherited forms of PD. The substitution of alanine to threonine at position 53 of the Alpha-synuclein protein (A53T) leads to a severe autosomal dominant trait of Parkinsonism, characterized by an early onset with a short disease duration to death [11,12].
There has been a strong link between the onset of PD with mitochondrial dysfunction and chronic inflammation. Many immune cell lineages including, T cells, mast cells, and macrophages play a key role in inflammation cascades affecting neural lineages and triggering neural degeneration [13]. Recently Haenseler et al. [14] generated iPSC derived macrophages from PD patients to confirm the impairment of normal homeostatic phagocytic function by alpha synuclein and attributing to this mechanism of SNCA in PD patients.
PGRN is a multifunctional growth factor widely expressed in various tissues with highest levels in epithelial and myeloid cells. PGRN is involved in cell proliferation, wound healing and modulation of inflammation [15]. Homozygous mutations in the progranulin gene (GRN) are associated with NCL, a rare lysosomal-storage disorder leading to accumulation of auto-fluorescent lipopigment in lysosomes detectable in various tissues, including skin, retina, and brain. Recently, homozygous GRN mutations were reported to be associated with behavioral variant frontal dementia and Parkinsonism. Heterozygous GRN mutations cause frontotemporal dementia (FTD) with transactive response DNA-binding protein of 43 kD (TDP-43)-positive inclusions [16]. There are no disease-modifying therapies for either FTD or NCL, in part because of a poor understanding of how mutations in GRN contribute to disease pathogenesis and neurodegeneration. This mutation also impairs immune cell types like macrophages. GRN−/− macrophages fail to clear intracellular bacteria due to impairments in autophagy mechanisms. The impairment of autophagy caused by GRN mutations in neurons and immune cells can translate to increased risk of neurodegenerative disease with time [17]. Hence generation of iPSCs derived macrophages harboring GRN mutation can be very valuable to study iPSCs-based disease modeling of modelling for NCL.
MeCP2 is a transcriptional repressor that binds to methylated CpG dinucleotides throughout the genome. Mutations in MeCP2 leading to a partial loss of function are best studied in the onset of Rett syndrome, a neurodevelopmental disorder that affects girls almost exclusively, and in some males [18]. Although Ret syndrome was originally diagnosed exclusively in females, with an approximate prevalence of 1 in 10,000 female births [19], and mutations in the MECP2 gene were previously thought to be fatal for males. A recent review by Volkumar et al. [20] summarized various case reports on the prevalence of MeCP2 mutations in males with Rett syndrome. They observed that nearly 65% of the patients exhibit other genetic co morbidities and hence miss out on the diagnosis for Rett syndrome. The authors emphasize the need to detect, evaluate and treat male patients harboring variant forms of MeCP2 and gather the necessary clinical information on these patient groups. Rett syndrome is characterized by normal early growth and development followed by a slowing of development, loss of purposeful use of the hands, distinctive hand movements, slowed brain and head growth, problems with walking, seizures, and intellectual disability. MeCP2 mutations affect multiple neural cell types like astrocytes, neurons and microglia. In addition to the impairment of function on many neural lineages, peripheral macrophage populations are also sensitive to alterations in MeCP2 expression on a cellular and molecular level highlighting the extensive crosstalk between the central nervous system and immune system with the onset of Rett syndrome [21]. Although Rett's syndrome is primarily studied in affected females, there are reported cases of Rett's syndrome in males that are severe and lethal [20,22]. Hence generation of iPSC-derived macrophages and other neural lineages mimicking the loss of function of MeCP2 can be very valuable to study iPSC-based disease modeling of Rett syndrome.
iPSC derived cell lineages are a useful source of patient-specific cells that otherwise are difficult or impossible to derive for disease modeling. In the present paper we have followed through on this approach to interrogate disease associated genotypes in a physiological context by first generating isogenic iPSCs containing a targeted gene mutation mimicking PD (SNCA A53T Homozygous (HO), NCL (GRN R493X Homozygous (HO)) and Rett Syndrome (MECP2 Hemizygous Truncation (HM) knockout (KO). Then further differentiating these iPSCs into functional, cryopreservable macrophages, along with an unengineered isogenic parental iPSC donor line as a reference. This panel of macrophages offers a unique toolset for many genotype-specific preclinical therapeutic discovery programs for neurodegenerative diseases. The present paper offers a unique tool set to study the role of peripheral macrophages engineered to express mutations associated typically seen in neuronal cells and understand the effects of these mutations in non-neural cell lineages and their contributions to enable in vitro disease modeling applications for neuro degenerative diseases.

Generation of iPSC lines
Multiple human iPSC lines were utilized in these experiments, including an apparently healthy normal (AHN) line and several engineered iPSC lines mimicking disease associated genotypes. The AHN line was the parent line for all engineering, allowing for isogenic models. The AHN human iPSC line (01279) was generated from male PBMCs. Using an episomal reprogramming method, a CD34+ enriched population was electroporated with an EBNA1/oriP based reprogramming vectors [23,24] then seeded onto RetroNectin-coated plates (Takara Bio, Inc; Otsu, Shiga Japan) and fed using a cocktail of small molecules before being transitioned to TeSR2 medium once iPSC colony formation was observed. The isolated colonies were then selected, propagated and cryopreserved for further characterization, genome engineering and differentiation experiments. This work was previously described by Mack et al. [25].
SNCA A53T HO (SNCA A53T) iPSCs were genetically engineered from AHN iPSC 01279 by nuclease-mediated homologous recombination and a donor oligo SJD 14-133. The resulting iPSCs contained SNP rs104893877 where amino acid 53 was changed from alanine to threonine resulting in the A53T variant in the alpha-synuclein gene (SNCA) as well as two silent mutations resulting in the SNCA A53T Homozygous iPSC line (SNCA A53T), provided a disease model for PD. GRN R493X HO (GRN R493X) iPSC was generated from AHN iPSC 01279 using nucleasemediated homologous recombination and a donor oligo SJD 14-180. The resulting GRN R493X iPSCs contained SNP rs63751294 changing amino acid 493 from arginine to a termination codon resulting in the early termination of the progranulin gene (GRN) as well as a three base change following the termination codon. The GRN R493X Homozygous diseased line (GRN R493X) provided a model for NCL.
Finally, AHN iPSC 01279 MeCP2 HM KO (MECP2 HM) KO line cell line was generated using nuclease-mediated homologous recombination and a donor plasmid p1553. Donor plasmid p1553 inserted a series of stop codons prior to the Methyl CpG Binding domain followed by a PGKp-PuromycinR-SV40pA selection cassette flanked by LoxP sites. The MECP2 HM KO line provided a disease model for Rett Syndrome.
The presence of HPCs were characterized by CD34 high /CD43 high /CD45 low , CD41 low / CD235a low , and CD34 high /CD31 high populations. Purification of HPCs was achieved using Indirect CD34 MicroBead Kit (Miltenyi Systems). The CD34 positive cells were cryopreserved in CryoStor10 (Biolife Solutions) using a Control Rate Freezer (CRF) and transferred to liquid nitrogen tanks for storage. Multipotency of HPCs derived from parental and isogenically engineered lines was confirmed by plating 5000 HPCs on serum free MethoCult (STEMCELL Technologies) media and quantifying colony-forming units per the manufacturer's instructions. The colonies were manually scored after 12 days on incubation. Individualized HPCs were also plated at 100,000 cells per chamber slides in the presence of serum free MegaCult1-C collagen-based medium (STEMCELL Technologies) to detect megakaryocyte progenitors. After 10 days Megacult cultures were dehydrated, fixed, and stained to detect the presence of Mk-specific antigen GPllb/llla (CD41) on megakaryocytes. All slides were processed according to the manufacturer's instructions.
Cryopreserved or live CD34 positive HPCs were differentiated in to macrophages according to the method described by Choi et al. [26,27] with slight modifications in the protocol. HPCs were first expanded to generate Common Myeloid Progenitors (CMPs) that were stained with CD34, CD43, and CD45 to demonstrate CD34 low /CD43 high /CD45 high population characterizing the emergence of CMPs. CMPs were further differentiated into macrophages using a serum free maturation medium in the presence of M-CSF and IL-1Beta (PeproTech). Cultures were maintained in a normoxic environment, fed every four days and sampled for emergence of CD68 positive cells. Cells were sampled and fixed using 4% PFA for 15 minutes and then permeabilized using saponin. The percentage of CD68 was quantified by flow cytometry. Once end stage cultures reached a high purity of �80% CD68 expression, cells were harvested and cryopreserved in CryoStor10 (BioLife Solutions). The vials were cryopreserved using a CRF and transferred to liquid nitrogen tanks for storage. The duration of differentiation from iPSC to end stage macrophages varied from 39-49 days.

Characterization of macrophages
Characterization of end stage macrophages derived from AHN 01279, and engineered SNCA A53T, GRN R493X, and MECP2 HM, was performed post-cryopreservation. Macrophages were characterized by cell surface expression of macrophage specific markers and presence of distinct morphology. Live and cryopreserved macrophages post thaw were assessed for cytokine release and phagocytosis function. Cryopreserved macrophages were thawed and placed in end stage macrophage maturation media post thaw for 1-3 days to enable staining of cell surface markers, stimulations, and phagocytosis functional assays. Live macrophage cultures were harvested and tested alongside cryopreserved macrophages for all functional assays.
For morphological assessment, macrophages were transferred to Shandon Double Cytoslides (Thermo Scientific) and stained using Wright Stain (Fisher Scientific).
Macrophages Live and cryopreserved macrophages were plated at 30,000 cells per well into 96 Ultra Low Attachment (ULA) plate (Corning) in cytokine free base media. Cryopreserved macrophages were placed in media and allowed to rest for three days. The macrophages were then stimulated using LPS (1μg/mL), LPS (1μg/mL) + IFN-Gamma (50ng/mL), IL-4 (50ng/mL) + IL-13 (50ng/mL), IL-10 (50ng/mL) + TGF-Beta (25ng/mL), and TGF-Beta (25ng/mL). Untreated wells were kept as controls. The supernatants were collected 24 hours after stimulation and frozen at -20˚C for later analysis. The analytes were quantified using a multi-plex Luminex kit and the results were acquired on a FLEXMAP 3D system (Luminex). Data analysis was carried out using the xPONENT Multiplex Assay Analysis software (Luminex).
The phagocytotic capability of the live and cryopreserved macrophages were measured to access functionality. Macrophages were plated at 15,000 cells per well in tissue culture (TC) 96 well plate (Corning) in SFD media supplemented with ExCyte (Millipore), 20ng/ml M-SCF (PeproTech) and 10 ng/mL IL-1Beta (PeproTech). After 24 hours the wells were treated with pHrodo labeled Red S. aureus bioparticles (Thermo Fisher) at 0.5 μg/well, placed into IncuCyte S3 and measured for red fluorescent intensity for 6 days. As the S. aureus bioparticles are internalized the pHrodo dye reacts with the decrease of pH resulting in increasing level of fluorescence. Phase and red fluorescence images were captured in the IncuCyte S3 every 2 hours for 6 days at 10x magnification. Phagocytosis was quantified using IncuCyte1 s3 software (v2019B), which applied a pre-defined analysis definition to remove background fluorescence, allowing accurate quantification of red fluorescence intensity.

Sequence analysis
Genomic DNA (gDNA) was extracted from the iPSC lines and the differentiated cells post thaw. The regions of interest were amplified and submitted to Functional Bioscience (Madison, WI) for sequencing. The resulting sequence data were aligned to the expected sequence maps using SeqMan Pro (DNAStar, Madison, WI).

Human macrophage preparation, RNA-Seq library preparation, sequencing, and data analysis
Briefly, parental and isogenically engineered iPSCs were differentiated to end stage macrophages expressing >80% CD68 positive cells. The cells were cryopreserved and stored in the presence of liquid nitrogen. RNA seq analysis was performed on macrophages three days post thaw maintained in the presence of macrophage medium. RNA was extracted with RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol. After extraction, the sample was incubated with Turbo DNase at 37˚C for 30 minutes and subsequently re-purified using RNeasy clean-up protocol.
RNA sequencing was performed on Illumina NovaSeq 6000 platform (Illumina Inc., San Diego, CA) at Novogene, China targeting at least 20 million paired-end reads with a read length of 150 bp. Reads were mapped to the Ensembl (GRCh38.p10) Homo sapiens genome using splice-aware alignment program, HISAT2, v.2.1.0. Differential gene expression analysis between the wild-type and mutant macrophage samples was carried out using Cuffdiff, v.2.2.1 program. Custom perl scripts were used to convert the Cuffdiff output files to compatible text files for visualization in TIBCO Spotfire software v.11.0.0. Visualization of the mapped reads was accomplished using the Integrative Genomic Viewer, v. 2.4.15 (http://www.broadinstitute. org/igv). The GO biological process functional enrichment analysis of the statistically significant DEGs obtained from the RNASeq analysis between the mutant MO and the WT was accomplished in R using ClusterProfiler R package (http://www.bioconductor.org/packages/ release/bioc/html/clusterProfiler.html). The hypergeometric distribution method was used to calculate the P-value. P<0.05 was used as the threshold for statistical significance. The results of ClusterProfiler enrich analysis were visualized via the enrichplot (https://github.com/ GuangchuangYu/enrichplot) and cnetplot package [28,29]. Transcription tracks were assembled utilizing Trinity v2.8.4 assembler using default settings was used to assemble the transcriptomes de novo for both the 01279 MO and MeCP2 HZ MO mutant lines. All the 01279 and mutant transcripts which matched the HG38 MeCP2 sequence were identified using BLAST. A multiple sequence alignment of the assembled transcripts showing the disrupted mutant exon region was performed using clustalW.

Results
Generation of integration-free iPSC lines from AHN donor and subsequent engineering strategies to generate isogenic iPSC lines expressing SNCA A53T, GRN R493X, and MeCP2 HM AHN iPSC line 01279 generated using episomal reprogramming methods previously described by Mack et al. [25] was used for generating all engineered isogenic lines. AHN 01279 iPSCs were transfected using nuclease-mediated homologous recombination to generate three lines, SNCA A53T (Fig 1A), and GRN R493X ( Fig 1B) and MECP2 HM ( Fig 1C). Nuclease design considerations included pseudogenes, repetitive sequences and potential "off-by-one" cut sites in the genome. The SNCA A53T and GRN R493X nuclease recognition sites were analyzed using TAGScan (https://ccg.epfl.ch/tagger/tagscan.html) [30]. Two off-by-one sites were identified for the SNCA A53T nuclease and one site for the GRN R493X nuclease. Sanger sequence across these sites was performed on the engineered iPSC lines to confirm the sequence at these  Table). MECP2 TAL design was analyzed by ZFN-site (https://ccg.epfl.ch/tagger/targetsearch.html [30]) there were no predicted off-target sites with 2 or fewer mismatches per half site. The selected iPSC clones and parental lines were maintained using Matrigel coated plates and E8 media under hypoxic conditions for at least 5 passages and then cryopreserved. The parental iPSC line and genetically engineered iPSC lines were characterized further by quantifying the high purity of pluripotency markers TRA-160, SSEA-4, CD90, SOX-2, OCT3/4 and Nanog expression by flow cytometry (Fig 1D1 and 1D2 and S2 Table). The iPSCs banks displayed a normal karyotype (Fig 1E1-1E4) by G-banding karyotype analysis. Each of these engineered iPSCs offered an opportunity to study the effect on of selected mutations on hematopoiesis and differentiation to macrophages.

Parental and isogenic engineered iPSCs can differentiate to Hematopoietic Progenitor Cells (HPCs) at varying efficiencies
Hematopoietic differentiation was initiated by harvesting the iPSC cultures and generating aggregates (3D) under defined serum free conditions. A schematic of the differentiation process is displayed in Fig 2A. The protocol was able to successfully generate HPC from parental and isogenically engineered lines. The key phenotypic markers observed during hematopoietic differentiation, which are absent on iPSC (S2B Fig), are CD43+/CD34+ cells representing HPCs, CD41+/CD235+ cells representing erythromegakaryocytic lineage, CD45+/CD43+ representing myeloid lineages. Parental and isogenically engineered iPSC lines were placed in hematopoietic differentiation for~13 days and the expression profile of all hematopoietic markers was quantified by flow cytometry. The expression profiles, of hematopoietic lineage specific markers are depicted in Fig 2B. Although all iPSC successfully differentiated to HPCs, differences were observed in the expression levels of many markers. MECP2 HM iPSC revealed the highest expression of HPC associated markers on day 13 of differentiation as compared to SNCA A53T and GRN R493X engineered iPSCs. MECP2 HM IPSC derived HPC revealed the highest expression of erythroid lineage specific markers, while SNCA A53T iPSC derived HPCs revealed the lowest levels of hematopoietic, erythroid and myeloid specific markers ( Fig 2C). The ability of HPCs to differentiate into megakaryocytes and platelets was determined by placing HPCs in a Collagen IV based CFU assay for megakaryocyte progenitors for 10 days. The colonies were stained for the presence of CD41 and confirmed the megakaryocytic potential of HPCs ( Fig 2D). The multipotency of HPCs was assessed using colonyforming unit (CFU) assays to assess the proliferation and differentiation of HPCs towards different downstream lineages. CFU-E (colony-forming unit-erythroid) and the BFU-E (burstforming unit-erythroid) scored for erythroid cells; CFU-G (colony-forming unit-granulocyte) CFU-M (colony-forming unit-macrophage), CFU-GM (colony-forming unit-granulocyte/ macrophage) scored for the myeloid cell type while the CFU-GEMM (colony-forming unitgranulocyte/erythroid/macrophage/megakaryocyte) scored for the presence of true multipotent colonies.
CFUs plates from 01279 (n = 32), SNCA A53T (n = 6), GRN R493X (n = 6), and MECP2 HM (n = 6) derived HPCs were manually scored and quantified (Fig 2E and S3 Table). The functional assessment of HPCs derived from parental and isogenically engineered iPSCs revealed the lowest number of immature BFU-E (burst forming unit-erythroid) units in MECP2 HM derived HPCs indicating that MeCP2 may have role in erythroid maturation. MECP2 HM iPSC derived HPCs also showed an increase in the granulocyte/monocyte population compared to the parental unengineered iPSCs. SNCA A53T iPSC derived HPCs revealed a lower CFU potential for all lineages compared to the parental iPSC derived HPCs while the CFU potential of GRN R493X iPSC derived HPCs remained comparable to the parental unengineered iPSCs (Fig 2E). This the first report describing the differences in hematopoietic potential in iPSC derived lines, SNCA A53T, GRN R493X and MECP2 HM HPCs.

Pure end stage macrophages derived from iPSCs from parental and isogenic engineered iPSCs
HPCs derived from parental and isogenically engineered-iPSCs were further differentiated to CMP ( Fig 3A) and phenotypically characterized by the by loss of CD34 (Fig 3B) with concomitant increase in a cell population co-expressing CD43 and CD45. The emerging CMPs retain the potential to generate multiple myeloid lineages (mast cells, dendritic cells, granulocytes) besides generating end stage macrophages. The CMPs were expanded for 8 days under normoxic conditions in a 3D culture format. The enrichment and maturation to end stage macrophages was driven by M-CSF and IL-1 beta [26]. An in-process purity assessment of end stage cultures was performed by quantification of CD68 expression. As the purity of CD68 positive cells increased to >80% the cultures were harvested and cryopreserved. End stage macrophages were successfully generated from parental and isogenically engineered iPSCs. The efficiency of generation of macrophages varied between iPSC lines. The parental 01279 cells revealed the highest efficiency of generating 1.74 macrophages per iPSC, followed by SNCA A53T and GRN R493X, while MECP2 HM revealed the lowest efficiency of generating 0.21 macrophages per iPSC (Fig 3C).

Cryopreserved macrophages derived from parental and isogenic engineered iPSCs exhibit features of unpolarized/naïve macrophages
The identity of iPSC-derived macrophages was established by the qualitative analysis of cellular morphology and quantification of relevant phenotypic markers by flow cytometry. Macrophages are distinguished from other types of myelomonocytic cells by the distinct morphology observed by Wright staining and staining for a panel of surface markers. Cryopreserved macrophages exhibited high viability and recovery post thaw (S3 Fig). Cryopreserved macrophages from parental and isogenically engineered iPSCs revealed the classic macrophage morphology by Wright staining (Fig 4A). Expression of CD68 post thaw was evaluated to determine purity of macrophages derived from parental and isogenically engineered lines (Fig 4B). Macrophages were also stained for an extended panel of phenotypic markers three days post thaw (Fig 4C and S4 Table). Pro-inflammatory (M1) macrophages, characterized by the secretion of pro-inflammatory cytokines, high expression of CD64, CD80, CD86 and low expression of CD206 are responsible for phagocytosis and ADCC killing. Anti-inflammatory (M2) macrophages, which are involved in tissue repair and provide regulatory signals, express high levels of CD206 and CD11b.
End stage macrophages from parental and engineered lines revealed high expression of myeloid specific markers, CD33 (Siglec-3), CD11c; a member of the leukointegrin family, which binds to complement fragment (iC3b), and pan macrophage sialomucin CD68, a member of the scavenger receptor supergene family. Cryopreserved iPSC-derived macrophages also expressed major histocompatibility complex class II (HLADR), co-stimulatory molecules CD80 and CD86 [31] that support antigen presentation role for macrophages, high affinity immunoglobulin gamma Fc receptor CD64 that plays a central role in macrophage antibodydependent cellular cytotoxicity (ADCC) and release of pro-inflammatory cytokines expressed on M1 macrophages. Cryopreserved IPSC-derived macrophages also constitutively expressed high levels of CD206 a MRC1, a C-type mannose receptor 1 involved in immune homeostasis by scavenging unwanted mannoglycoproteins and CD11b, a β2 integrin involved in adhesion, which binds to inactivated completement 3b, expressed on M2 macrophages and microglia [32]. iPSC derived macrophages expressed high levels of MHC class I expression and low levels of CD1a expression which could aid in immune recognition and antigen presentation function. IPSC-derived macrophages also expressed CD169; a marker of a subpopulation of macrophages found in lymphoid organs and implicated in immune tolerance and antigen presentation [33]. Parental and isogenically engineered macrophages did not have detectable expression of human dendritic cell specific marker CD209 (DC-SIGN). The combined expression of both M1 and M2 specific markers indicate an uncommitted / M0 status of iPSCderived macrophages.
Although end stage macrophages derived from SNCA A53T, GRN R493X and MECP2 HM iPSCs expressed high levels of CD68, there were differences in the levels of expression of additional panel of markers. Comparative levels of expression of M1 (CD11c, CD64, CD80, CD86, and HLA-DR), M2 (CD206 and CD11b) and tissue specific regulatory macrophages (CD169) were observed in parental, SNCA A53T and GRN R493X iPSC derived macrophages. MECP2 HM iPSC derived macrophages revealed significantly lower level of expression of CD33, CD64, CD11b, CD206, CD86 compared to the parental macrophages.
Parental and SNCA A53T, GRN R493X and MECP2 HM macrophages expressed high levels of CD47, 'marker-of-self' protein, is emerging as a novel potent macrophage immune checkpoint for cancer immunotherapy [34]. Parental and SNCA A53T, GRN R493X and MECP2 HM macrophages also expressed high levels of fractalkine receptor CX3CR1, that plays a key role in macrophage maturation, wound healing, and orchestrating immune response [35,36]. This is the first report presenting confirming the expression of CD47 and CX3CR1 on iPSC derived macrophages implying a role in evading immune response.
Macrophages express CD38 under inflammatory conditions [37]. The absence of CD38 expression on parental, SNCA A53T, GRN R493X and MECP2 HM macrophages confirms the naïve and non-inflammatory status of end stage iPSC derived macrophages. Parental, SNCA A53T, GRN R493X and MECP2 HM macrophages also expressed low levels of Triggering receptor expressed on myeloid cells-2 (TREM-2) a transmembrane immune receptor. The low expression of TREM2 aids to distinguish iPSC derived macrophages from iPSC derived microglia, which express very high levels of TREM2 (

Phagocytic function of macrophages is retained pre and post cryopreservation
Phagocytosis is a key feature of macrophage function in host defense and tissue homeostasis. Quantification of macrophage phagocytosis in vitro has been recently made possible utilizing the IncuCyte S3 real time imaging platform and pHrodo labeled pathogen bioparticles, which only fluoresce when localized in the acidic environment of the phagolysosome [39]. Though most of functional analysis has been demonstrated with live cells we were keen to test this function post cryopreservation of end stage macrophages. End stage live and cryopreserved macrophages derived from parental and isogenic engineered iPSCs were able to phagocytose pHrodo labelled S. aureus, but there were differences in the kinetics of phagocytosis between lines, fresh and cryopreserved. Live end stage macrophages derived from the parental iPSC revealed a stronger intensity of phagocytosis when benchmarked against cryopreserved end stage macrophages derived from the same parental iPSC (Fig 5). This phenomenon has been reported by other investigators [40] in normal healthy macrophages as a response to cryopreservation process. On the other hand, cryopreserved macrophages from engineered iPSCs revealed a higher intensity of phagocytosis post thaw than live end stage macrophages derived from the same engineered iPSCs (Fig 5).
Side by side comparison of all live end stage macrophages cultures derived from SNCA A53T iPSC, GRN R493X iPSC, and MECP2 HM iPSC revealed a strong impairment of phagocytic function when compared to live end stage parental iPSC derived macrophages (S4 Fig). This observation supports data published by Haenseler et al. [14] on the impairment in phagocytic functions in live iPSC-derived macrophages from PD patients with SNCA Triplication and A53T mutations. Additionally, Wang et al. [41] reported an enhanced recruitment of live macrophages with the decreased bacterial clearance, impaired endocytosis capacity of macrophages accompanied with Progranulin (PGRN) deficiency and finally, Cronk et al. [42] reported on the differential gene expression profile on live Mecp2-null peritoneal macrophages implying a key role of this gene in sustaining macrophage function [42].
On the other hand, phagocytosis from cryopreserved macrophages generated from SNCA A53T iPSCs revealed a robust phagocytosis when compared to the parental unengineered macrophages. GRN R493X iPSC derived macrophages displayed a similar kinetic trend of A possible explanation to support this observation is the bias towards an M1 sub type in engineered macrophages before cryopreservation due to the high secretion levels of the pro inflammatory cytokines IL-8, CCL11, and CCL17 when compared to the unstimulated parental macrophages in end stage live cultures (Fig 5). The bias towards an M1 polarization could influence metabolic reprogramming in engineered macrophages which in turn could trigger interplay between metabolic enzymes and metabolites of different pathways and contribute to enhanced phagocytosis function in engineered macrophages post cryopreservation [43].
To analyze the cytokine profile in parental and engineered macrophages, end stage live and cryopreserved macrophages three days post thaw were stimulated with LPS, LPS + interferon, IL-4 + IL-13, IL-10 + TGF-beta, and TGF-beta. The supernatants were examined for the production of key cytokines using the Luminex Multiple assay system (S5-S8 Figs). The fold induction of the analytes in the live engineered macrophages was measured as a fold induction with respect to the analytes released by live parental and engineered macrophages. Similarly, the fold induction of the analytes in the cryopreserved engineered macrophages was measured as a fold induction with respect to the analytes released by cryopreserved parental un engineered macrophages.
Unstimulated parental and engineered iPSC-derived macrophages released similar levels of IL-23, MMP-9, CCL3, CD163, CCL22, and CXCL1 (Fig 6). Unstimulated engineered macrophages had a higher secretion of IL-8, CCL11, and CCL17; and lower levels of IL-1ra, CCL24, and fractalkine compared to the unstimulated parental macrophages. Unstimulated engineered iPSC derived macrophages also spontaneously produced CCL1 in cultures. CCL1 is involved in inflammatory processes through leukocyte recruitment and could play a crucial role in calcium flux, angiogenesis and along with CCL2 and CCL9 are negative regulators of M2 polarization neuroinflammatory disorders [48][49][50].
Live and cryopreserved macrophages were plated in cytokine free media and cytokine release was quantified per stimulant. Cryopreserved macrophages were thawed and allowed to recover for 72 hours prior to stimulation. Macrophages were stimulated with LPS, LPS + Interferon Gamma, Il-4 + IL-13, IL-10 + TGF-Beta, and TGF-Beta for 24 hours. Supernatants were collected, and the release of cytokines measured by Luminex multiplex system (n = ± SE). Heat maps capture the fold induction of each analyte released by engineered lines over the analytes released by parental line in the presence the aforementioned stimulants. White color represented no change red color represented a higher release of cytokines, and blue color demonstrated a decrease in cytokine release in the engineered lines as compared to AHN parental line.

RNAseq analysis investigates transcriptional similarities and differences between cryopreserved parental and engineered iPSC derived macrophages
We used RNA-seq analysis to investigate transcriptional similarities and differences between parental vs engineered iPSC-derived macrophages to understand differences in transcription profile and uncover underlying mechanisms that might be triggered by perturbations in SNCA, Progranulin, and MeCP2 genes. Transcription tracks were generated from end stage macrophages confirming presence of genetic engineering at transcription level (S9 Fig). The high purity of end stage macrophage cultures (routinely 90-95% pure) confirmed the absence of contaminating cell types to interfere with the analysis. The RNA seq data revealed an upregulation of 431 genes and concomitant downregulation of 276 in SNCA A53T Macrophages, an upregulation of 549 genes and concomitant downregulation of 356 in GRN R493X macrophages, an upregulation of 466 genes and concomitant downregulation of 406 genes in MeCP2HM macrophages when compared to the parental unengineered macrophages (Fig 8). The cumulative analysis of the gene expression profile of all three engineered lines revealed an overlapping set of 70 genes (Fig 8) in common between all three engineered lines in comparison to the parent line. The complete list of all 70 gene are included in S5A-S5C Table in the Supporting Information. The profile of each engineered line is highlighted in a separate table. The overall expression profile indicates a down regulation of 50 genes in the engineered macrophages reflecting impairment of function and an increase in a subset of genes that regulate migration, metabolism and proliferation all key to survival of the engineered lines. RNA-Seq analysis on the dysregulated genes in engineered macrophages indicated a twothreefold upregulation of CMTM1, ATRNL1, NFKBIZ, MCOLN2, P2RY6, PLEKHG5, GATM and ADGRE4P genes when compared to the unengineered parental macrophages. MTM1 belongs to chemokine-like factor gene superfamily, MCOLN2 gene that plays a role in chemokine secretion, macrophage migration and the regulation of innate immune response and finally ADGRE4P that play a role in adhesion and migration function of macrophages. The upregulation of these genes implied an increased secretion of chemokines facilitating adhesion and migration function in engineered macrophages. The engineered cells also revealed a twothreefold upregulation of ATRNL1 gene that regulates energy homeostasis and GATM gene that affects mitochondrial enzymes supporting the hypothesis for an increase or shift in metabolism post thaw in engineered macrophages. The increase in the levels of NFKBIZ, one of the nuclear I kappa B proteins augments IL-6 production and mis regulation of signaling of the MAPK Erk Pathway implying a bias to the M1 subtype accompanied by an altered cell proliferation kinetics in the engineered lines. An upregulation of ACER2 promotes cell proliferation and survival in engineered lines.
MECP2 HM macrophages revealed a decrease in the expression profiles of FBP1, SPRED1, GATM, ZNF366 COL1A2, TNFSF15, SLC9A9, MPEG1, MARCH1, SEMA3A genes whereas as SNCA A53T macrophages and GRN R493X macrophages displayed and increase in expression of all the above-mentioned genes. A closer look at the gene functions indicated their role in sugar uptake, mitochondrial metabolism, activators of pathways affecting lysosomal and vesicular transport and structural integrity of cells. The downregulation of these genes and the 50 additional genes supports the poor function and analyte profile seen in MECP2 HM macrophages. The gene concept network plots offer a deeper visual analysis to the linkages of genes and biological concepts as a network (Fig 9) to highlight the pathways affected in engineered macrophages regulating cell metabolism, exocytosis, transport, degranulation, immune activation, vascular circulation and neural differentiation pathways.

Discussion
Macrophages are crucial orchestrators of both initiation and resolution of immune responses, they play a key role in maintaining homeostasis in most organs of the body. Inflammation is associated with several neurogenerative diseases [4]. Presence of self-maintaining Peripheral Nervous System-resident macrophages that share gene expression patterns with microglia have been reported in aging and neurodegenerative conditions. Farina et al. [53] reported a phenotypic convergence between microglia and peripheral macrophages under pathological conditions suggesting a synergy dictated by the microenvironment. Peripheral macrophages have been reported to act as mediators enhancing the expression of Parkinson's Disease (PD) related genes, such as LRRK2 [9] or show impairment in phagocytosis of amyloid beta in AD patients with ApoE4/4 genotype [7]. Peripheral macrophages can also play a reverse role in calming chronic inflammation and delay the progression of neurodegenerative diseases by reacting to the stimulus like Niacin and polarization to the M2 subtype [8]. This novel information about the biology of microglia and peripheral macrophages sheds new light about their therapeutic potential for neuroinflammatory and neurodegenerative diseases.
The present study describes the development of a defined, scalable differentiation protocol to enable generation of large batches of cryopreserved end stage macrophages from AHN iPSCs as well as isogenically engineered variants that are implicated in various neurological disorders. The method consistently generates a phenotypically pure population of functional end stage macrophages extensively characterized by phenotypic markers. End stage live and cryopreserved macrophages retained viability, purity and phagocytic function. The functional assessment of SNCA A53T, GRN R493X, and MECP2 HM macrophages on eliciting an M1 or M2 dependent responses post stimulation was quantified between live and cryopreserved macrophages from parental and engineered iPSCs. RNA-seq analysis was used to investigate transcriptional similarities and differences between parental and engineered macrophages and the mechanisms that might be underlying the observed differences between parental and engineered macrophages that effect immune responses and function. The results highlight the effect of site-specific genetic alterations in SNCA at A53T and GRN at R493X on the development and temporal relationships between upregulation of genes and secretion of cytokines/chemokines favoring macrophage polarization towards the inflammatory M1 subtype, thus providing new insights into the role of macrophages in neurodegenerative diseases. The data also sheds light on the functional impairment of MECP2 HM macrophages and the subtle differences in functional performance of SNCA A53T and GRN R493X iPSC derived macrophages. Further experimentation is needed to understand the striking changes in metabolism and the interplay between different pathways adapted by macrophages to survive and function post cryopreservation. These findings contribute to understand the effects of perturbation of MeCP2, A53T and GRN at R493X on non-neural lineages. In summary, our study confirms the utility of iPSC-derived macrophages genetically engineered to mimic disease specific mutations a valuable in vitro model to study the role of innate immune system in the onset of neurodegenerative diseases.